The applicant's long range goal is to develop methods to investigate the mechanism by which the alphavirus replication complex is assembled and the rate of transcription is regulated. Her aims in the current proposal are 1. To use genetic analysis to probe the functions of nonstructural proteins in overall replication and in the conversion form minus strand to plus strand synthesis. Conditional lethal mutants will be studied to identify the role of the nsP3 phosphorylation in RNA replication, the second-site changes in nsP or RNA sequences rescuing mutants in the NTPase/helicase domain of nsP2, and the basis of homologous interference by alphavirus nsP mutants. 2. To use an in vitro reconstitution system to investigate temperature sensitive defects in promoter recognition, initiation, elongation or conversion of initial replication complexes (RC-initial) to stable replication complexes (RC-stable) of specific nsP1-4 mutants. 3. To purify and characterize replication complexes from infected cells and to identify essential host factors.